PROJECT SUMMARY/ABSTRACT MLL-rearranged (MLL-r) leukemias account for 5-10% of human acute leukemia and is associated with poor prognosis. The unmet clinical needs and the lack of an effective targeted therapy to the MLL-r leukemias emphasize the need for novel regimens. Recent cancer epigenetics studies discovered a central role for the histone H3 lysine 79 (H3K79) methyltransferase DOT1L in MLL-r leukemogenesis. Important clinical responses have been noted with DOT1L inhibitor treatment as a single agent, however, it is expected that combination treatments will be necessary. Our preliminary studies based on a DOT1L-inhibitor sensitization screen have identified an essential role of the PHF20/KAT8 histone acetyltransferase complex, in supporting the expression of DOT1L-driven oncogenes. The objective of this application is to determine the critical epigenetic mechanisms that collaborate with DOT1L to maintain oncogene expression in MLL-r leukemia. Our central hypothesis is that PHF20 mediates KAT8 recruitment to maintain the locus-specific histone acetylation and transcription of the DOT1L-driven leukemic program. We will investigate the efficacy of DOT1L and PHF20/KAT8 combination therapies (Aim 1), dissect the PHF20/KAT8 chromatin targeting mechanisms (Aim 2), and validate a novel high-density CRISPR protein scan technology for de novo discovery of the functional elements in DOT1L/PHF20/KAT8 (Aim 3). This study is innovative because (1) it introduces a novel concept of simultaneously targeting multiple components of an epigenetic feed-forward loop to efficiently suppress the cancer programs, and (2) it establishes a brand new genetic screen approach for a sub-protein level functional domain discovery. The impact of this research will be of significance because (1) it immediately provides novel therapeutic opportunities against the difficult-to-treat MLL-r leukemias, and (2) it will help identify novel functional elements in epigenetic regulators for future pharmaceutical targeting.